Citrus Tristeza Virus (CTV) is a serious pathogen in most citrus producing regions of the world. It has the largest RNA genome of any plant virus known. There are many known strains of CTV, each of which can cause a variety of symptoms in different host species. These known strains of CTV have been categorized into five major groups based on their biological activity. These groups are commonly known as: mild; seedling yellows (SY); decline on sour orange (QD); stem pitting on grapefruit (SP-G); and stem pitting on sweet orange (SP-0) (Garnsey, S. M., Barrett, H. C., and Hutchison, D. J., 1987. "Identification of citrus tristeza virus resistance in citrus relatives and potential applications." Phytophylactica 19:187-197). The symptoms caused by the different strains of CTV can range from insignificant, to disfiguring, to severe damage even of mature trees. Mild strains cause no symptoms on most commercial and commonly grown citrus varieties; whereas the strains in the other four categories cause severe effects including, for example, death of the host citrus trees.
Such a great diversity of symptoms caused by CTV requires techniques to detect and differentiate these strains if CTV is to be controlled. Recognizing this need, the State of Florida, which benefits greatly from the citrus industry, implemented the Quality Tree Program of Florida, which mandates frequent testing of thousands of citrus propagation trees for the presence of severe strains of CTV. Symptoms development on differential indicator hosts (McClean et al., 1977, Garnsey, supra) and aphid transmissibility (Roistacher, C. N. and Bar-Joseph, M., 1984. "Transmission of tristeza and seedling yellows tristeza by small population of Aphis gossypii." Plant Disease 68:494-496) were used for this purpose in the past.
Since these methods were costly and time-consuming, other techniques such as cDNA probe hybridization (Rosener, A., Lee, R. F., and Bar-Joseph, M. 1986. "Differential hybridization with cloned cDNA sequences for detecting a specific strain of citrus tristeza virus." Phytopathology 76:820-824), dsRNA analysis (Dodds, J. A., R. J. Jordan, Roistacher, C. N., and T. Jarupat. 1987. "Diversity of citrus tristeza virus isolates indicated by dsRNA analysis." Intervirology 27:177-188), polypeptide map analysis (Guerri, J., Moreno, P., and Lee R. F. 1990. "Identification of citrus tristeza virus strains by peptide maps of virion coat protein." Phytopathology 80:692-698) and RFLP analysis (Gillings et al., 1993; Akbulut, 1995) were developed. Hybridization using nucleic acid probes, either radioactively or nonradioactively labeled, has been used for detection and/or differentiation of many plant pathogens, including the viruses CTV (Rosner, supra), potato virus Y (Singh et al 1995), geminiviruses (Gilbertson et al., 1991) luteoviruses (Martin et al., 1990) and insect transmitted viruses (Harper and Creamer 1995). Although these methods enabled differentiation of certain strains of CTV, they are not adaptable to rapid large scale assays.
As an alternative to these methods, a monoclonal antibody, MCA13, was developed, which reacts predominantly with most severe strains, but not with mild strains of CTV (Permar, T. A., Garnsey, S. M., Gumpf, D. J., and Lee, R. L. 1990. "A monoclonal antibody that discriminate strains of citrus tristeza virus." Phytopathology 80:224-228). The current method of analysis for the presence or absence of CTV employs MCA13 in an enzyme-linked immunosorbent assay (ELISA) which has the advantage of being scaled up to process large numbers of samples at a relatively low cost. It has been determined that the MCA13 binds to a particular epitope of the capsid protein of CTV. Although the ELISA method using MCA13 is sensitive and reliable, it can only differentiate between mild and severe strains of CTV. There currently is no method available to differentiate between and amongst the mild and severe strains such as QD, SP-0, and SP-G.
Accordingly, a nucleic acid probe which can differentiate between the severity of symptoms caused by strains of CTV in plants is highly desirable as a diagnostic tool for detecting the potential problems of CTV infection in a plant. In addition, a nucleic acid probe having a further advantage of being capable of being scaled up to process large numbers of samples would be beneficial to programs such as the Florida Quality Tree Program, and the citrus industry as a whole.